Characterization of non-standard viral genomes during arenavirus infections identifies prominent S RNA intergenic region deletions.

Arenaviruses, a family of negative-sense RNA viruses spread by rodents, are a leading cause of severe hemorrhagic fever in humans. Due to a paucity of antivirals and vaccines for arenaviruses, there is a need to identify new mechanisms for interfering with arenavirus replication. In several negative-sense RNA viruses, natural viral interference results from the production of non-standard viral genomes (nsVGs) that activate the innate immune system and/or compete for essential viral products. Although it is well established that arenaviruses produce strong interfering activities, it is unknown if they produce interfering nsVGs. Here, we show that arenaviruses produce deletions within the intergenic region of their small (S) RNA genome, and these deletions inhibit viral glycoprotein production during minigenome replication. S RNA deletions are more abundant when arenaviruses are grown in high-interfering conditions and are associated with reduced viral replication. Overall, we found that arenaviruses produce internal deletions within the S RNA intergenic region that are capable of decreasing glycoprotein production. These natural arenavirus interfering molecules provide a new target for the generation of therapeutics against arenaviruses.IMPORTANCEArenaviruses are hemorrhagic fever-causing pathogens that infect millions of people a year. There are currently no approved antivirals that target arenaviruses, and understanding natural mechanisms that inhibit arenavirus replication is crucial for the development of effective therapeutics. Here, we identified multiple deletions within arenavirus genomes that remove major replicative elements of the viral genomes. We show that deletions that remove the intergenic region of the viral genome can prevent viral protein production. These deletions were found in all arenaviruses tested in this study representing a mechanism that could be harnessed for the development of antivirals that broadly target the arenavirus family.